The long-term goal of this research is to elucidate the mechanisms involved in the regulation, development, and function of muscarinic acetylcholine receptors (mAChR) in the embryonic chick heart. The chick embryo represents an attractive system for the study of cardiac mAChR both in vivo and in cell culture. While the basic functional properties of mAChR in chick and mammalian heart are similar, they exhibit distinct pharmacological, immunological, and biochemical properties. We have previously isolated two distinct receptor genes expressed in chick heart, and have evidence using the polymerase chain reaction that at least one additional subtype is present. This proposal will isolate clones encoding the remainder of the mAChR subtypes expressed in chick heart and determine their biochemical and functional properties. Previous work has demonstrated that the number and function of both mAChR and G-proteins are regulated in tissue-specific manner in chick heart during embryonic development. This proposal will use subtype-specific nucleic acid and antibody probes to test the hypothesis that changes in specific mAChR subtypes of mAChR-G-protein interactions are responsible for these developmentally regulated changes. We have found that incubation of chick heart cells in culture with agonists induces a decrease not only in the levels of mAChR polypeptide but also a decrease in mRNAs encoding both receptor subtypes cloned to date. This proposal will determine the mechanism responsible for the regulation of mAChR mRNA. Previous whole animal studies have suggested that the number and function of mAChR can be regulated by insulin and thyroid hormone. We have used a serum-free defined medium culture system to demonstrate that insulin regulates the number of mAChR binding sites, the level of mRNA encoding only one of the two mAChR subtypes, and the functional sensitivity of the mAChR, and have found that thyroid hormone can regulate the number of mAChR binding sites. This proposal will determine the mechanisms and functional consequences for the regulation of mAChR expression by insulin and thyroid hormone. This research will provide valuable new information the basic mechanisms regulating the expression and function of mAChR in the heart. In addition, this research may aid in understanding the etiology of a variety of cardiac abnormalities.